Name: Video: Transfection and Harvesting Adeno-Associated Virus Vectors Using HEK293T Cells for Gene Therapy
Uploaded: 2024-05-03T14:20:00
Duration: 4 min
Description: 264 Views. Transfection and Harvesting Adeno-Associated Virus Vectors Using HEK293T Cells for Gene Therapy

transfection and harvesting adeno-associated virus vectors using hek293t cells for gene therapy

Efficient Adeno-Associated Virus Vector Production Using a Cell Factory Platform

Adeno-associated virus (AAV) vectors have become an indispensable tool in gene therapy research, offering a unique combination of efficacy and safety for gene delivery1. Traditional methods for generating AAVs in laboratory settings have been pivotal in advancing our understanding and application of gene therapy2. However, these methods, while foundational, exhibit certain limitations and challenges, especially in terms of yield, time efficiency, and the quality of the vectors produced, notably the ratio of full to empty particles3.
The conventional procedure for AAV production primarily involves the transfection of HEK293 cells4. This process, typically conducted in cell culture dishes, requires the cells to be transfected with a plasmid containing the gene of interest along with helper plasmid&#160;and AAV capsid plasmid5,6. Following transfection, the cells produce AAV particles, which are then harvested and purified5,6. The purification process often involves ultracentrifugation, a critical step in obtaining high-purity AAV vectors7. Ultracentrifugation, particularly using cesium chloride (CsCl) or iodixanol gradient, is a standard method for separating AAV particles from cellular debris and other impurities8. This step is crucial for achieving the desired purity and concentration of AAV vectors, which directly impacts their efficacy in gene delivery8. Despite its widespread use, traditional ultracentrifugation has its drawbacks. For example, the yield of AAV vectors from this method can be variable and often low, which poses significant challenges when large quantities of high-titer vectors are needed, particularly for in vivo studies or large animal models9.
Another critical aspect of AAV vector quality is the ratio of full to empty particles10. AAV preparations often contain a mixture of these particles; however, only the full particles contain the therapeutic genetic material. The presence of a high proportion of empty particles can significantly reduce the efficiency of gene delivery10. Assessing and optimizing the ratio of full to empty particles is thus a key parameter in evaluating the efficacy of AAV vectors. Traditional methods, while capable of producing AAV vectors, often struggle to control this ratio consistently, leading to variations in vector potency10.
Here, a unique method is presented, which streamlines the process of generating high-yield and high-purity AAV vectors using a cell factory platform free from using labor-intensive HEK293T cell cultures in cell dishes, integrating polyethylene glycol/aqueous two-phase partitioning with iodixanol gradient ultracentrifugation. The AAV vector purity is confirmed via SDS-PAGE and silver staining, and the full-to-empty particle ratio is determined using transmission electron microscopy (TEM), meeting the quality standards for in vivo studies11.

Author Spotlight: Advancing Gene Therapy Research with High-Titer Adeno-Associated Virus Vector Production

A Streamlined and Standardized Procedure for Generating High-Titer, High-Quality Adeno-Associated Virus Vectors Utilizing a Cell Factory Platform

Transfection and Harvesting Adeno-Associated Virus Vectors Using HEK293T Cells for Gene Therapy

transfection-harvesting-adeno-associated-virus-vectors-using-hek293t

Research

JoVE Journal

Bioengineering

264 Views. Transfection and Harvesting Adeno-Associated Virus Vectors Using HEK293T Cells for Gene Therapy

Video: Transfection and Harvesting Adeno-Associated Virus Vectors Using HEK293T Cells for Gene Therapy

Preclinical gene therapy research, particularly in rodent and large animal models, necessitates the production of AAV vectors with high yield and purity. Traditional approaches in research laboratories often involve extensive use of cell culture dishes to cultivate HEK293T cells, a process that can be both laborious and problematic. Here, a unique in-house method is presented, which simplifies this process with a specific cell factory (or cell stacks, CF10) platform. An integration of polyethylene glycol/aqueous two-phase partitioning with iodixanol gradient ultracentrifugation improves both the yield and purity of the generated AAV vectors. The purity of the AAV vectors is verified through SDS-PAGE and silver staining, while the ratio of full to empty particles is determined using transmission electron microscopy (TEM). This approach offers an efficient cell factory platform for the production of AAV vectors at high yields, coupled with an improved purification method to meet the quality demands for in vivo studies.

As the field of gene therapy continues to evolve, there is a growing need for innovative methods that can address these challenges. Here, a unique method is presented, which streamlines the process of generating high-yield and high-purity AAV vectors using a cell factory platform, meeting the quality standards for in vivo studies.

Preclinical gene therapy research, particularly in rodent and large animal models, necessitates the production of AAV vectors with high yield and purity. ...